Treatment of chronic inflammatory conditions

ABSTRACT

A method for the treatment of a chronic inflammatory condition in a patient which comprises administration to the patient of an agent which blocks or inhibits IL-3 signalling events in the patient.

FIELD OF THE INVENTION

This invention relates generally to the treatment of chronic inflammatory conditions, and in particular to a method for reducing or otherwise ameliorating the effects of such conditions in patients. In one particular embodiment, the present invention relates to the treatment of arthritis, particularly rheumatoid arthritis which is a chronic, inflammatory disease characterised by the presence of numerous inflammatory mediators and by the destruction of diarthrodial joints.

BACKGROUND OF THE INVENTION

Interleukin-3 (IL-3) is a cytokine that can improve the body's natural response to disease as part of the immune system. IL-3 stimulates the differentiation of multipotent hematopoietic stem cells (pluripotent) into myeloid progenitor cells as well as stimulating proliferation of all cells in the myeloid lineage (erythrocytes, thrombocytes, granulocytes, monocytes, and dendritic cells). It is secreted by activated T cells to support growth and differentiation of T cells from the bone marrow in an immune response.

IL-3 exerts its activity through binding to a specific cell surface receptor known as the Interleukin-3 receptor (IL-3 R). IL-3 R is a heterodimeric structure composed of a 70 kDa IL-3 R alpha (CD123) and a 120-140 kDa IL-3 R beta (CD131). The IL-3 R alpha chain (IL-3Rα) has a very short intracellular domain while the IL-3 R beta chain (IL-3Rβ) has a very large cytoplasmic domain. IL-3 R alpha binds IL-3 with relatively low affinity. In the presence of IL-3 R beta, however, IL-3 R alpha has a much higher affinity for IL-3. It is not clear how signal transduction occurs following IL-3 binding, however recent studies suggest signalling requires formation of a higher order complex comprising a dodecamer¹. The IL-3 R beta chain is also shared by the receptors for IL-5 and GM-CSF. Cells known to express IL-3 receptors include hematopoietic progenitors, mast cells, basophils and blood monocytes as well as more mature cells of various hematopoietic lineages including monocytes, macrophages, neutrophils, basophils, mast cells, eosinophils, megakaryocytes, erythroid cells, and CD5⁺ B cell sub-populations². Non-hematopoietic cells have also been shown to express the receptor including some endothelial cells, stromal cells, dendritic cells and Leydig cells^(2,3).

IL-3 provides a potentially important connection between the immune and hemopoietic systems⁴. It may be important for the production and function of mast cells and basophils particularly during immune reactions⁵. IL-3 can also promote the growth and activation of macrophage lineage populations⁶⁻⁸ and can help to generate dendritic cells⁹. As noted above, IL-3 signalling is mediated by a common receptor beta-subunit (IL-3Rβ) and a specific ligand-binding alpha-subunit (IL-3Rα), although in the mouse there is an additional beta-subunit¹⁰.

Very little is known regarding the role of IL-3 in chronic inflammatory conditions, such as rheumatoid arthritis (RA). IL-3 mRNA could not be detected in the synovium of RA patients in one study¹¹ but was found in a later study¹²; some but not all RA patients have been found to have detectable IL-3 in the circulation¹³ and there is an association between a single-nucleotide polymorphism in the IL-3 gene promoter and RA¹⁴. However, IL-3 levels decrease during arthritis progression in a rat arthritis model¹⁵ and it has been reported recently that IL-3 administration inhibits murine inflammatory arthritis¹⁶.

In work leading to the present invention, the inventors have found that the effects of chronic inflammatory conditions such as RA can be inhibited or reduced by blocking or interfering with the ligand/receptor interaction between IL-3 and IL-3R. This finding is quite surprising and was unexpected in view of the recent report¹⁶ suggesting that IL-3 administration has the potential to diminish the inflammatory response and indirectly arrest cartilage and bone loss in inflammatory arthritis.

Bibliographic details of the publications referred to in this specification are referenced at the end of the description.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for the treatment of a chronic inflammatory condition in a patient, which comprises administration to the patient of an agent which blocks or inhibits IL-3 signalling events in the patient.

In another aspect, the invention provides the use of an agent which blocks or inhibits IL-3 signalling events in, or in the manufacture of a medicament for, the treatment of a chronic inflammatory condition in a patient.

In yet another aspect, the invention provides an agent for the treatment of a chronic inflammatory condition in a patient, wherein said agent blocks or inhibits IL-3 signalling events in the patient.

In other aspects of this invention, the agent may be formulated in a pharmaceutical composition together with one or more pharmaceutically acceptable excipients and/or diluents, or it may be provided in a kit which optionally includes instructions to use the agent in accordance with a method for the treatment of a chronic inflammatory condition in a patient.

The chronic inflammatory condition may be, for example, arthritis, more particularly inflammatory arthritis such as RA.

Preferably, the agent is one which blocks or inhibits IL-3/IL-3R or IL-3/IL-3R alpha interactions in the patient. Preferably also the patient is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows collagen-induced arthritis (CIA) progression (clinical score) in mice receiving anti-IL-3 mAb or PBS (control). Results expressed as mean±SEM. n=10 mice treated group, n=6 mice control group.

FIG. 2 shows effect of IL-3 signalling on the cell viability of monocytes (CD14—derived from PBMC). The monocytes were plated at a density of ˜1.8×10⁶ cells per 6 cm IWAKI (low adherence) TC dish and cultured for 7 days in RPMI+10% FCS and: IL-3 (3 ng/ml) alone, IL-3 (3 ng/ml)+IL-3-R antibody, or IL-3 (0.3 ng/ml) alone, or IL-3 (0.3 ng/ml)+IL-3-R antibody. IL-3-R antibody was used at 1 μg/ml in each case. At day 4 new IL-3 and IL-3-R antibodies were added. On day 7 cells were removed and counted.

FIG. 3 shows IL-3 induces the basophil activation marker CD203c in a dose-dependent manner. PBMC from urticaria patients (URT: URT001, URT002, URT004, URT005, URT10, URT11) and a normal donor (NOR) were isolated and were stimulated with polyclonal IgE, FMLP, FMLP and IL-3 or an increasing concentration of IL-3. The percentage of CD203+ve basophils were calculated after staining with an antibody cocktail to identify basophils and with an antibody against the basophil activation marker CD203c then analyzing by flow cytometry.

FIG. 4 shows anti-IL-3R antibody blocks IL-3-induced basophil activation. PBMC from a normal donor were isolated and stimulated with an increasing concentration of IL-3 in the presence or absence of a neutralizing anti-IL-3R antibody (CSL360). The percentage of CD203+ve basophils were calculated after staining with an antibody cocktail to identify basophils and with an antibody against the basophil activation marker CD203c then analyzing by flow cytometry.

FIG. 5 shows anti-CD123 monoclonal antibody (CSL362) depletes basophil in a time-dependent manner. PBMC from a normal donor were isolated and incubated without antibody or with a depleting anti-CD123 antibody (CSL362) for various times (as indicated: 4 hrs., 24 hrs., 48 hrs., 72 hrs.). The percentage of basophils remaining were calculated after staining with an antibody cocktail to identify basophils and analyzing by flow cytometry.

FIG. 6 shows anti-CD123 mAb reproducibly depletes basophils within 24 hr. PBMC from three normal donors were isolated and incubated without antibody or with a depleting anti-CD123 antibody (CSL362) for 24 h. The percentage of basophils remaining were calculated after staining with an antibody cocktail to identify basophils and analyzing by flow cytometry.

FIG. 7 shows anti-CD123 mAb activates NK cells within 24 hr. PBMC from three normal donors (NOR006, NOR008, NOR009) were isolated and incubated without antibody (solid line) or with a depleting anti-CD123 antibody (CSL362, dashed line) for 24 h. Activation of NK cells (CD56+ve cells) was determined by CD16 down-regulation. PBMC were stained with anti-CD56 antibodies to identify NK cells and with anti-CD16 antibodies. Loss of CD16 staining, as compared with the no antibody control, was determined by flow cytometry.

FIGS. 8( a) and (b) show depletion of murine basophils in peripheral blood by in vivo administration of anti-IL-3Rα antibodies. BALB/c mice were intravenously administered with an anti-IL-3Rα antibody (1C2), anti-CD200R3 (Ba103) or the isotype control antibodies mouse IgG2a (mIgG2a) or rat IgG2b (rIgG2b). All antibodies were injected at 30 μg per mouse except for 1C2 which was injected at 18 μg per mouse. Peripheral blood cells were isolated 24 h post antibody administration and stained for FcεR1α and CD49b expression to identify basophils. Representative staining profiles from flow cytometry analysis are shown in FIG. 8( a). Basophil populations are boxed. The percentage of basophils per mouse were calculated from flow cytometry analysis and the mean (+SEM) of 3 mice per group are shown in FIG. 8 (b). ***: p<0.001, **:p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method for the treatment of a chronic inflammatory condition in a patient, which comprises administration to the patient of an agent which blocks or inhibits IL-3 signalling events in the patient.

Preferably, the agent is one which blocks or inhibits IL-3/IL-3R or IL-3/IL-3R alpha interactions in the patient.

Chronic inflammatory conditions which may be treated in accordance with the present invention are well known to persons skilled in this field, and include in particular arthritis, more particularly inflammatory arthritis such as adult and juvenile RA. Other indications include but are not limited to chronic obstructive pulmonary disease (COPD); inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; chronic inflammatory demyelinating polyneuropathy (CIDP); atherosclerosis; scleroderma; systemic lupus erythematosus (SLE); Sjogren's syndrome; gout; osteoarthritis; polymyalgia rheumatica; seronegative spondyloarthropathies including ankylosing spondylitis; Reiter's disease, psoriatic arthritis, mixed connective tissue disease (MCTD); chronic Lyme arthritis; Still's disease; chronic urticaria; uveitis associated with rheumatoid arthritis and disorders resulting in inflammation of the voluntary muscle and other muscles, including dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleiomyomatosis.

Reference herein to “treatment” is to be considered in its broadest context and includes both therapeutic treatment and prophylactic or preventative measures. Patients in need of treatment include those already afflicted with a chronic inflammatory condition as well as those in which such a condition is to be prevented. Patients who are partially or totally recovered from the condition might also be in need of treatment. The term “treatment” does not necessarily imply that a patient is treated until total recovery. Accordingly, treatment includes reduction or amelioration of the symptoms of a particular chronic inflammatory condition as well as halting or at least retarding the onset, development or progress of, reducing the severity of, or eliminating, a particular chronic inflammatory condition.

The agent which is administered in accordance with the present invention blocks or inhibits the activation of IL-3 signalling events in the patient, preferably by blocking or inhibiting IL-3/IL-3R or IL-3/IL-3R alpha interactions. As used herein, a reference to “blocks or inhibits IL-3 signalling events in the patient” encompasses any intervention which leads to a decreased level of IL-3 initiated signalling. Such interventions include, by way of example, the use of agents which specifically block or inhibit the activation of IL-3 signalling events (e.g. agents which target IL-3, IL-3Rα, IL-3Rβ), as well as the use of agents designated to selectively target cells capable of IL-3 signalling and by such targeting induce cell death (e.g. agents which target IL-3R and carry an anti-cellular moiety).

In one embodiment of the invention, the agent may be an antigen binding molecule which binds selectively to IL-3, or to IL-3R, the IL-3R alpha or the IL-3R beta.

As used herein the term “antigen binding molecule” refers to an intact immunoglobulin, including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, or to antigen-binding (including, for example, Fv, Fab, Fab′ and F(ab′)₂ fragments) and/or variable-domain-comprising fragments of an immunoglobulin that compete with the intact immunoglobulin for specific binding to the binding partner of the immunoglobulin, e.g. a host cell protein. Regardless of structure, the antigen-binding fragments bind with the same antigen that is recognized by the intact immunoglobulin. Antigen-binding fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or they may be genetically engineered by recombinant DNA techniques. The methods of production of antigen binding molecules and fragments thereof are well known in the art and are described, for example, in Antibodies, A Laboratory Manual, Edited by E. Harlow and D. Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which is incorporated herein by reference.

Preferably, the antigen binding molecule is a monoclonal antibody.

In this embodiment of the invention, the antigen binding molecule may comprise a modified Fc region, more particularly a Fc region which has been modified to provide enhanced effector functions, such as enhanced binding affinity to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). For the IgG class of antibodies, these effector functions are governed by engagement of the Fc region with a family of receptors referred to as the Fcγ receptors (FcγRs) which are expressed on a variety of immune cells. Formation of the Fc/FcγR complex recruits these cells to sites of bound antigen, typically resulting in signalling and subsequent immune responses. Methods for optimizing the binding affinity of the FcγRs to the antibody Fc region in order to enhance the effector functions, in particular to alter the ADCC and/or CDC activity relative to the “parent” Fc region, are well known to persons skilled in the art, and are described, for example, in International Patent Publication No. WO 2009/070844. These methods can include modification of the Fc region of the antibody to enhance its interaction with relevant Fc receptors and increase its potential to facilitate ADCC and ADCP. Enhancements in ADCC activity have also been described following the modification of the oligosaccharide covalently attached to IgG1 antibodies at the conserved Asn²⁹⁷ in the Fc region.

The term “binds selectively”, as used herein, in reference to the interaction of an antigen binding molecule, e.g. an antibody or antibody fragment, and its binding partner, e.g. an antigen, means that the interaction is dependent upon the presence of a particular structure, e.g. an antigenic determinant or epitope, on the binding partner. In other words, the antibody or antibody fragment preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms.

Without wishing to be bound by any particular theory, it is believed that in this embodiment of the invention, by binding selectively to IL-3, or to IL-3R, IL-3R alpha or IL-3R beta, an antigen binding molecule such as an antibody or antibody fragment blocks or inhibits the ligand/receptor interaction and thereby interferes with IL-3 signal activation.

In one embodiment, the antigen binding molecule may be a monoclonal antibody which binds selectively to IL-3R alpha (CD123). Thus, the antigen binding molecule may be monoclonal antibody (MAb) 7G3, raised against CD123, which has previously been shown to inhibit IL-3 mediated proliferation and activation of both leukaemic cell lines and primary cells (see U.S. Pat. No. 6,177,678 to Lopez). Alternatively, the agent may be the monoclonal antibody CSL360, a chimeric antibody obtained by grafting the light variable and heavy variable regions of the mouse monoclonal antibody 7G3 onto a human IgG1 constant region (see International Patent Publication No. WO 2009/070844). Like 7G3, CSL360 binds to CD123 (human IL-3Rα) with high affinity, competes with IL-3 for binding to the receptor and blocks its biological activities. CSL360 also has the advantage of potential utility as a human therapeutic agent by virtue of its human IgG1 Fc region which would be able to initiate effector activity in a human setting. Moreover, it is likely that in humans it would show reduced clearance relative to the mouse 7G3 equivalent and be less likely to be immunogenic. Further examples of this antigen binding molecule include humanised antibody variants of 7G3 or CSL360, fully human anti-CD123 antibodies and anti-CD123 antibodies with enhanced effector function (such as ADCC activity) as described, for example in Example 4 of International Patent Publication No. WO2009/070844.

In another embodiment of the present invention, the agent may be an IL-3 mutein which binds to IL-3R but either does not lead to or at least results in reduced IL-3 signal activation. Generally, these ‘IL-3 muteins’ include natural or artificial mutants differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues. An example of an IL-3 mutein which binds to IL-3R but exhibits reduced IL-3 signal activation is a 16/84 C→A mutant¹⁷. IL-3 muteins may also include modified polypeptides in which one or more residues are modified to, for example, increase their in vivo half life. This could be achieved by attaching other elements such as a PEG group. Methods for the PEGylation of polypeptides are well known in the art.

In another embodiment of the present invention, the agent is a soluble receptor which is capable of binding to IL-3. Examples of such soluble receptors include the extracellular portion of IL-3R alpha or a fusion protein comprising the extracellular portion of IL-3R alpha fused to the extracellular portion of IL-3R beta.

In yet another embodiment, the agent may comprise an anti-cellular moiety that can be targeted to cells capable of IL-3 signalling to induce cell death. In some embodiments, the agent may comprise the anti-cellular moiety conjugated to an antigen binding molecule which binds selectively to IL-3, or to IL-3R, IL-3R alpha or IL-3R beta. In other embodiments the agent may comprise an anticellular moiety conjugated to a IL-3 mutein. Examples of suitable anti-cellular moieties include chemotherapeutics, radioisotopes or cytotoxins. Chemotherapeutics include a hormone such as a steroid; an anti-metabolite such as cytosine arabinoside, fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycin C; a vinca alkaloid; demecolcine; etoposide; mithramycin; calicheamycin, CC-1065 and derivatives thereof, or an alkylating agent such as chlorambucil or melphalan, a coagulant, a cytokine, growth factor, bacterial endotoxin or the lipid A moiety of bacterial endotoxin. Radioisotopes include α-emitters such as, for example, 211Astatine, 212Bismuth and 213Bismuth, as well as β-emitters such as, for example, 131Iodine, 90Yttrium, 177Lutetium, 153Samarium and 109Palladium, and Auger emitters such as, for example, 111Indium. Cytotoxins include generally a plant-, fungus-or bacteria-derived toxin, such as an A chain toxin, a ribosome inactivating protein, a-sarcin, aspergillin, restirictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention just a few examples, as well as cytotoxins derived from marine organisms such as sponges, such as Kahalalide F, Ecteinascidin (Yondelis™), or Variolin B, for example.

The agent is administered in an effective amount. An “effective amount” means an amount necessary at least partly to attain the desired response or to delay or inhibit progression or halt altogether, the progression of the particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the racial background of the individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. If necessary, the administration of the agent may be repeated one or several times. The actual amount administered will be determined both by the nature of the condition which is being treated and by the rate at which the agent is being administered.

Preferably, the patient is a human, however the present invention extends to treatment and/or prophylaxis of other mammalian patients including primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals.

In accordance with the present invention, the agent is preferably administered to a patient by a parenteral route of administration. Parenteral administration includes any route of administration that is not through the alimentary canal (that is, not enteral), including administration by injection, infusion and the like. Administration by injection includes, by way of example, into a vein (intravenous), an artery (intraarterial), a muscle (intramuscular) and under the skin (subcutaneous). The agent may also be administered in a depot or slow release formulation, for example, subcutaneously, intradermally or intramuscularly, in a dosage which is sufficient to obtain the desired pharmacological effect.

In another aspect, the present invention provides the use of an agent which blocks or inhibits IL-3 signalling events in, or in the manufacture of a medicament for, the treatment of a chronic inflammatory condition in a patient.

In yet another aspect, the present invention provides an agent for the treatment of a chronic inflammatory condition in a patient, wherein said agent blocks or inhibits IL-3 signalling events in the patient.

In this aspect of the invention, the agent as described above may be formulated in a pharmaceutical composition together with one or more pharmaceutically acceptable excipients and/or diluents.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol and lactic acid. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, suitable carbohydrates (e.g. sucrose, maltose, trehalose, glucose) and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The formulation of such therapeutic compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

In a further aspect of the invention, there is provided a kit comprising (i) an agent as described above, and optionally (ii) instructions to use the agent in accordance with a method for the treatment of a chronic inflammatory condition in a patient.

The present invention is further illustrated by the following non-limiting Examples.

Example 1

The effect of a neutralizing monoclonal antibody (mAb) to murine IL-3 on disease progression was tested in the collagen-induced arthritis (CIA) model, the most widely used murine model for rheumatoid arthritis.

Male DBA/1 mice (8-12 weeks old, 10 mice per group) were immunized intradermally with type II collagen in adjuvant on days 0 and 21¹⁸.

Mice were assessed for redness and swelling of limbs and a clinical score was allocated for each limb using an established scoring system as follows: 0—normal; 1—slight swelling and/or erythema; 2—extensive swelling and/or erythema; 3—severe swelling; 4—severe swelling and/or rigidity. Severity of arthritis is expressed in terms of the mean clinical score totalled for all four limbs (range 0-16 per mouse).

Mice were treated with 250 μg anti-IL-3 mAb (Southern Biotech)/mouse or PBS on day 21, 23, 25, 28 and 30. As can be seen in FIG. 1, there was a suppression of disease severity in the anti-IL-3-treated group.

This experiment is particularly encouraging as in this particular experiment there was a very rapid induction of a very severe disease with the plateau in the control mice being reached quickly. This plateau is normally reached about one week later.

The literature data for the relatively acute murine CIA indicates that neutralizing anti-IL-3 Ab is required soon after disease induction and is ineffective if delayed post disease onset¹⁹. This does not necessarily mean that IL-3 would not be a target in inflammation. We do not yet understand the background inflammatory/autoimmune events responsible for driving diseases such as rheumatoid arthritis (RA) and how closely these events are mirrored in animal models. Even for a chronic condition such as RA there are patients with “acute onset” disease. Also, anti-IL-3 therapy might still be beneficial in RA, for example, since the disease often relapses providing opportunities to suppress its exacerbations. It should also be borne in mind that the commonly used anti-inflammatory glucocorticoids are widely believed to act by down-regulating inflammatory mediator gene expression at the transcriptional level—they do not work in vitro in surrogate inflammation assays if added subsequent to the inciting stimulus.

Example 2

The role of IL-3 signalling as a pro-inflammatory cytokine in CD14+ monocytes was investigated. Peripheral Blood Mononuclear Cells (PBMC) were isolated from a Red Cross donor buffy pack. CD14+ monocytes were then purified by negative selection (MACS separation) such that approximately 80% of the cells were CD14+ as assessed by flow cytometry. Monocytes were plated at a density of ˜1.8×10⁶ cells per 6 cm IWAKI (low adherence) TC dish and cultured for 7 days in RPMI+10% FCS and: IL-3 (3 ng/ml) alone, IL-3 (3 ng/ml)+IL-3-R antibody, IL-3 (0.3 ng/ml) alone, or IL-3 (0.3 ng/ml)+IL-3-R antibody. IL-3-R antibody was used at 1 ug/ml in each case. At day 4 new IL-3 and IL-3-R antibodies were added. On day 7 cells were removed and counted before being lysed in RNA lysis buffer. The results indicate that IL-3 provides a pro-survival stimulus for the monocytes in a dose dependent manner and this effect was overcome by anti-IL-3R antibody (FIG. 2). Thus blocking or inhibiting IL-3 signalling in CD14+ monocytes prevents their survival and accumulation at sites of inflammation and as such provides a means to control inflammation.

Example 3

Urticaria, commonly known as hives, is an inflammatory condition that manifests as recurrent wheals that range in size from several centimeters down to just a few millimeters. Generally the wheals are pink in color with a pale center and can be associated with a burning or prickly sensation. Urticaria can present at any age and 1%-5% of the population will present with urticaria at some point in their lifetime. Chronic urticaria greatly impacts on quality of life, similar to patients with severe atopic dermatitis, psoriasis or acne. Most cases of chronic urticaria are idiopathic in nature; however, it is becoming increasingly clear that in many cases (35%-50%) auto-antibodies to the high affinity IgE receptor (FcεR1) or IgE itself are present, suggesting that chronic urticaria may be an autoimmune disease. Mast cells and basophils are the main cell types that express FcεR1 and respond to auto-antibodies in urticaria patients. When activated, mast cells and basophils release large amounts of histamine, which is the main effector molecule driving urticarial wheal formation. Both mast cells and basophils express the IL-3 receptor and IL-3 can prime these cells to produce increased levels of effector molecules, such as histamine, when exposed to triggers such as IgE and C5a.

PBMC from seven urticaria patient samples and four normal donor samples have been analyzed. IL-3 can activate human basophils ex vivo (FIG. 3) and a neutralizing anti-IL-3Rα antibody (CSL360—see International Patent Publication No. WO2009/070844) can inhibit IL-3 induced human basophil activation (FIG. 4). An ADCC optimized anti-IL-3Rα antibody (CSL362—an afucosylated variant of humanized and affinity matured anti-CD123 mAb 168-26 as described in International Patent Publication No. WO 2009/070844) can deplete human basophils from PBMC in a time dependent manner (FIG. 5). Complete or near-complete basophil depletion was observed in three independent donors within 24 hours of CSL362 addition (FIG. 6). NK cell activation was observed within 24 hours of addition of CSL362 suggesting that the depletion of basophils is via NK-cell mediated ADCC (FIG. 7).

Example 4

This example demonstrates that anti-IL-3R antibodies can also affect the number and level of activation of basophils in vivo. Female BALB/c mice (10-12 weeks old) were treated with a tail vein intravenous injection of anti-IL3Rα antibody 1C2 (see Example 5) or the control mouse IgG2a. A commercial antibody Ba103 that targets against CD200R3 and has been shown to specifically deplete mouse basophils²⁰, and its control antibody rat IgG2b were tested alongside as the controls. All antibodies were injected at 30 μg in 200 μl of PBS with the exception of 1C2 which was administered at 18 μg. A day later, peripheral blood and peritoneal cells were isolated and single suspension of cells prepared. Cells were pre-incubated with anti-FcγRII-III to prevent nonspecific binding. Cells were stained with FITC-conjugated anti-FcεRIα monoclonal antibody and PE-conjugated anti-CD49b monoclonal antibody to identify basophils (FcεRIα⁺ CD49b⁺). FITC-hamster IgG and PE-rat IgM were used as the isotype controls for FcεRIα and CD49b antibodies, respectively. Debris was gated out using a forward scatter (FSC) versus side scatter (SSC) and dead cells were discriminated with 7-amino-actinomycin D (7-AAD) staining. Stained cells were then analysed with FACSCanto™ (BD Biosciences) and data analysed using FlowJo software.

A single intravenous injection of 18 μg anti-IL3Rα antibody 1C2 induced a drastic reduction in basophil frequency in peripheral blood, to approximately 23% of the level in the isotype control (mouse IgG2a)-treated mice (FIG. 8). This depletion efficacy was comparable to that from the administration of 30 μg Ba103. Unlike the effect on basophils, no significant reduction in the frequency of peritoneal mast cells was noted in the mice treated with anti-IL3Rα or Ba103 antibodies (data not shown). The observation with Ba103 antibody on mast cells is consistent with the studies by Obata and colleagues²⁰.

Example 5

1) Generation of murine IL3 receptor specific monoclonal antibodies Antibody sequences that specifically recognised murine IL-3R alpha (mIL3Rα) were isolated from a library of human antibody sequences expressed as Fab fragments fused to the gIII protein on the surface of the filamentous bacteriophage M13 (Dyax Corp.). Anti-mIL3Rα phage-displayed antibody fragments were isolated by incubation of the phage library with a commercially-available purified recombinant fusion protein consisting of amino acids 17-331 of mIL3Rα fused to residues 100-330 of human IgG1 (Fc fragment) with a short polypeptide linker (sequence: IEGRID) supplied by R&D systems Inc. Specifically-bound phage were enriched and isolated as individual clones using standard methods. Individual clones were tested for specific binding to both the original target (mIL3Rα-Fc fusion) and to the extracellular domain of mIL3Rα expressed as residues 1-331 with a C-terminal hexa-histidine tag (msIL-3R-6His). Phage clones that bound to these targets and not to control proteins were selected for further analysis. Unique clones were identified by DNA sequencing of both polypeptide chains of the encoded antibody and the binding affinity of these clones for mIL-3R-6His was quantified by competitive ELISA. Clones with acceptable affinity were selected for re-engineering and expression as chimeric antibodies (human variable regions and murine IgG2a/kappa constant regions) for further analysis.

2) Mammalian expression vector construction for transient expression Heavy and light chain variable regions for the mIL3Rα-specific antibodies were PCR amplified from the phagemid vectors using standard molecular biology techniques. The heavy chain variable region was then cloned into the mammalian expression vector pcDNA3.1(+)-mIgG2a, which is based on the pcDNA3.1(+) expression vector (Invitrogen) modified to include the murine IgG2a constant region and a terminal stop codon. The light chain variable region was cloned into the expression vector pcDNA3.1(+)-mx, which is based on the pcDNA3.1(+) expression vector modified to include the murine kappa constant region. The expression vectors also contained a Kozak translation initiation sequence, an ATG start codon and appropriate signal peptides.

3) Cell Culture

Serum-free suspension adapted 293-T cells were obtained from Genechoice Inc. Cells were cultured in FreeStyle™ Expression Medium (Invitrogen) supplemented with penicillin/streptomycin/fungizone reagent (Invitrogen). Prior to transfection the cells were maintained at 37° C. in humidified incubators with an atmosphere of 8% CO2.

4) Transient Transfection

Transient transfection of the anti-mIL3Rα expression plasmids using 293-T cells was performed using 293fectin transfection reagent (Invitrogen) according to the manufacturer's instructions. The light and heavy chain expression vectors were combined and co-transfected with the 293-T cells. Cells (1000 ml) were transfected at a final concentration of 1×106 viable cells/ml and incubated in a Cellbag 2 L (Wave Biotech/GE Healthcare) for 5 days at 37° C. with an atmosphere of 8% CO2 on a 2/10 Wave Bioreactor system 2/10 or 20/50 (Wave Biotech/GE Healthcare). The culture conditions were 35 rocks per minute with an angle of 8°. Pluronic® F-68 (Invitrogen), to a final concentration of 0.1% v/v, was added 4 hours post-transfection. 24 hours post-transfection the cell cultures were supplemented with Tryptone N1 (Organotechnie, France) to a final concentration of 0.5% v/v. The cell culture supernatants were harvested by centrifugation at 2500 rpm and were then passed through a 0.45 μM filter (Nalgene) prior to purification.

5) Analysis of Protein Expression

After 5 days 20 μl of culture supernatant was electrophoresed on a 4-20% Tris-Glycine SDS polyacrylamide gel and the antibody was visualised by staining with Coomassie Blue reagent.

6) Antibody Purification

Anti-mIL3Rα antibodies were purified using protein A affinity chromatography at 4° C., where MabSelect resin (5 ml, GE Healthcare, UK) was packed into a 30 ml Poly-Prep empty column (Bio-Rad, CA). The resin was first washed with 10 column volumes of pyrogen free GIBCO Distilled Water (Invitrogen, CA) to remove storage ethanol and then equilibrated with 5 column volumes of pyrogen free phosphate buffered saline (PBS) (GIBCO PBS, Invitrogen, CA). The filtered conditioned cell culture media (1 L) was loaded onto the resin by gravity feed. The resin was then washed with 5 column volumes of pyrogen free PBS to remove non-specific proteins. The bound antibody was eluted with 2 column volumes of 0.1M glycine pH 2.8 (Sigma, MO) into a fraction containing 0.2 column volumes of 2M Tris-HCl pH 8.0 (Sigma, MO) to neutralise the low pH. The eluted antibody was dialysed for 18 hrs at 4° C. in a 12 ml Slide-A-Lyzer cassette MW cutoff 3.5 kD (Pierce, IL) against 5 L PBS. The antibody concentration was determined by measuring the absorbance at 280 nm using an Ultraspec 3000 (GE Healthcare, UK) spectrophotometer. The purity of the antibody was analysed by SDS-PAGE, were 2 μg protein in reducing Sample Buffer (Invitrogen, CA) was loaded onto a Novex 10-20% Tris Glycine Gel (Invitrogen, CA) and a constant voltage of 150V was applied for 90 minutes in an XCell SureLock Mini-Cell (Invitrogen, CA) with Tris Glycine SDS running buffer before being visualised using Coomassie Stain, as per the manufacturer's instructions.

REFERENCES

-   1. Hansen G, Hercus T R, McClure B J, Stomski F C, Dottore M, Powell     J, Ramshaw H, Woodcock J M, Xu Y, Guthridge M, McKinstry W J, Lopez     A F, Parker M W. The structure of the GM-CSF receptor complex     reveals a distinct mode of cytokine receptor activation. Cell. 2008;     134(3):496-507. -   2. Moretti S, Lanza F, Dabusti M., Tieghi A, Campioni D, Dominici M,     Castoldi G L. CD123 (Interleukin 3 receptor a chain). J Biol Regul     Homeost Agents 2001; 15 98-100. -   3. Yamada M, Suzu S, Tanaka-Douzono M, Wakimoto N, Hatake K,     Hayasawa H, Motoyoshi K. Effect of cytokines on the     proliferation/differentiation of stroma-initiating cells. J Cell     Physiol. 2000; 184:351-355. -   4. Schrader J W. In: Thomson A W, Thotze M, eds. The Cytokine     Handbook (ed 2nd). London; 2003: 201-225. -   5. Lantz C S, Boesiger J, Song C H, et al. Role for interleukin-3 in     mast-cell and basophil development and in immunity to parasites.     Nature. 1998; 392:90-93. -   6. Koike K, Stanley E R, Ihle J N, Ogawa M. Macrophage colony     formation supported by purified CSF-1 and/or interleukin 3 in     serum-free culture: evidence for hierarchical difference in     macrophage colony-forming cells. Blood. 1986; 67:859-864. -   7. Cheung D L, Hamilton J A. Regulation of human monocyte DNA     synthesis by colony-stimulating factors, cytokines, and cyclic     adenosine monophosphate. Blood. 1992; 79:1972-1981. -   8. Hart P H, Whitty G A, Burgess D R, Hamilton J A. Regulation by     interleukin-3 of human monocyte pro-inflammatory mediators.     Similarities with granulocyte-macrophage colony-stimulating factor.     Immunology. 1990; 71:76-82. -   9. Ebner S, Hofer S, Nguyen V A, et al. A novel role for IL-3: human     monocytes cultured in the presence of IL-3 and IL-4 differentiate     into dendritic cells that produce less IL-12 and shift Th cell     responses toward a Th2 cytokine pattern. J Immunol. 2002;     168:6199-6207. -   10. Metcalf D, Nicola N A. The hemopoietic colony-stimulating     factors: from biology to clinical applications. Cambridge:     University Press; 1995. -   11. Firestein G S, Xu W D, Townsend K, et al. Cytokines in chronic     inflammatory arthritis. I. Failure to detect T cell lymphokines     (interleukin 2 and interleukin 3) and presence of macrophage     colony-stimulating factor (CSF-1) and a novel mast cell growth     factor in rheumatoid synovitis. J Exp Med. 1988; 168:1573-1586. -   12. Heller R A, Schena M, Chai A, et al. Discovery and analysis of     inflammatory disease-related genes using cDNA microarrays. Proc Natl     Acad Sci USA. 1997; 94:2150-2155. -   13. Ferraccioli G, Falleti E, De Vita S, et al. Circulating levels     of interleukin 10 and other cytokines in rheumatoid arthritis     treated with cyclosporin A or combination therapy. J Rheumatol.     1998; 25:1874-1879. -   14. Yamada R, Tanaka T, Unoki M, et al. Association between a     single-nucleotide polymorphism in the promoter of the human     interleukin-3 gene and rheumatoid arthritis in Japanese patients,     and maximum-likelihood estimation of combinatorial effect that two     genetic loci have on susceptibility to the disease. Am J Hum Genet.     2001; 68:674-685. -   15. Lee J C, Dimartino M J, Votta B J, Hanna N. Effect of auranofin     treatment on aberrant splenic interleukin production in adjuvant     arthritic rats. J Immunol. 1987; 139:3268-3274. -   16. Yogesha S D, Khapli S M, Srivastava R K, et al. IL-3 inhibits     TNF-alpha-induced bone resorption and prevents inflammatory     arthritis. J Immunol. 2009; 182:361-370. -   17. L C Dorssers, M C Mostert, H Burger, C Janssen, P J Lemson, R     van Lambalgen, G Wagemaker and R W van Leen. Receptor and antibody     interactions of human interleukin-3 characterized by mutational     analysis. J. Biol. Chem., 1991, Vol. 266, 21310-21317. -   18. Cook A D, Braine E L, Campbell I K, Rich M J, Hamilton J A.     Blockade of collagen-induced arthritis post-onset by antibody to     granulocyte-macrophage colony-stimulating factor (GM-CSF):     requirement for GM-CSF in the effector phase of disease. Arthritis     Res. 2001; 3:293-298. -   19. Brühl H, Cihak J, Niedermeier M, Denzel A, Rodriguez Gomez M,     Talke Y, Goebel N, Plachý J, Stangassinger M, Mack M. Important role     of interleukin-3 in the early phase of collagen-induced arthritis.     Arthritis Rheum. 2009; 60(5):1352-1361. -   20. Obata K, Mukai K, Tsujimura Y et al. Basophils are essential     initiators of a novel type of chronic allergic inflammation. Blood.     2007; 110:913-920. 

1. A method for the treatment of a chronic inflammatory condition in a patient in need thereof, which comprises administration to the patient of an agent which blocks or inhibits IL-3 signalling events in the patient.
 2. The method according to claim 1, wherein the agent blocks or inhibits IL-3/IL-3R or IL-3/IL-3R alpha interactions in the patient.
 3. The method according to claim 1, wherein the agent is an antigen binding molecule which binds selectively to IL-3, or to IL-3R, IL-3R alpha or IL-3R beta.
 4. The method according to claim 3, wherein the antigen binding molecule is a monoclonal antibody, or an antigen-binding and/or variable-domain-comprising fragment thereof.
 5. The method according to claim 4, wherein the antigen binding molecule is a monoclonal antibody which binds selectively to IL-3.
 6. The method according to claim 4, wherein the antigen binding molecule is a monoclonal antibody which binds selectively to IL-3R alpha.
 7. The method according to claim 1, wherein the agent comprises an anti-cellular moiety.
 8. The method according to claim 7, wherein the anti-cellular moiety is a radioisotope selected from the group consisting of an α-emitter, a β-emitter, and an Auger emitter.
 9. The method according to claim 1, wherein the agent is an IL-3 mutein which binds IL-3R but does not lead to IL-3 signal activation.
 10. The method according to claim 1, wherein the agent is a soluble receptor which is capable of binding to IL-3.
 11. The method according to claim 10, wherein the agent is an extracellular portion of IL-3R alpha or a fusion polypeptide comprising an extracellular portion of IL-3R alpha fused to an extracellular portion of IL-3R beta.
 12. The method according to claim 1, wherein the chronic inflammatory condition is arthritis.
 13. The method according to claim 12, wherein the chronic inflammatory condition is rheumatoid arthritis (RA).
 14. The method according to claim 1, wherein the patient is a human.
 15. Use of an agent which blocks or inhibits IL-3 signalling events in, or in the manufacture of a medicament for, the treatment of a chronic inflammatory condition in a patient. 16.-25. (canceled)
 26. An agent for the treatment of a chronic inflammatory condition in a patient, wherein said agent blocks or inhibits IL-3 signalling events in the patient.
 27. The pharmaceutical composition according to claim 37, wherein the agent blocks or inhibits IL-3/IL-3R or IL-3/IL-3R alpha interactions in the patient.
 28. The pharmaceutical composition according to claim 37, wherein the agent is an antigen binding molecule which binds selectively to IL-3, or to IL-3R, IL-3R alpha or IL-3R beta.
 29. The pharmaceutical composition according to claim 28, wherein the antigen binding molecule is a monoclonal antibody, or an antigen-binding and/or variable-domain-comprising fragment thereof
 30. The pharmaceutical composition according to claim 29, wherein the antigen binding molecule is a monoclonal antibody which binds selectively to IL-3.
 31. The pharmaceutical composition according to claim 29, wherein the antigen binding molecule is a monoclonal antibody which binds selectively to IL-3R alpha.
 32. The pharmaceutical composition according to claim 37, wherein the agent comprises an anti-cellular moiety.
 33. The pharmaceutical composition according to claim 32, wherein the anti-cellular moiety is a radioisotope selected from the group consisting of an α-emitter, a β-emitter, and an Auger emitter.
 34. The pharmaceutical composition according to claim 37, wherein the agent is an IL-3 mutein which binds IL-3R but does not lead to IL-3 signal activation.
 35. The pharmaceutical composition according to claim 37, wherein the agent is a soluble receptor which is capable of binding to IL-3.
 36. The pharmaceutical composition according to claim 35, wherein the agent is an extracellular portion of IL-3R alpha or a fusion polypeptide comprising an extracellular portion of IL-3R alpha fused to an extracellular portion of IL-3R beta.
 37. A pharmaceutical composition comprising an agent for the treatment of a chronic inflammatory condition in a patient, wherein said agent blocks or inhibits IL-3 signalling events in the patient, together with one or more pharmaceutically acceptable excipients and/or diluents.
 38. A kit comprising (i) an agent for the treatment of a chronic inflammatory condition in a patient, wherein said agent blocks or inhibits IL-3 signalling events in the patient, and optionally (ii) instructions to use said agent in accordance with a method for the treatment of a chronic inflammatory condition in a patient in need thereof.
 39. The method according to claim 7, wherein the anti-cellular moiety is a radioisotope selected from 211Astatine and 131Iodine.
 40. The method according to claim 1, wherein the chronic inflammatory condition is inflammatory arthritis.
 41. The pharmaceutical composition according to claim 33, wherein the anti-cellular moiety is a radioisotope selected from 211Astatine and 131Iodine. 